Dual effect of the novel peptide antagonist K-14585 on proteinase-activated receptor-2-mediated signalling

Background and purpose:

Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl}-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)₂-mediated intracellular signalling events.

Experimental approach:

Using NCTC2544 cells expressing PAR₂, we assessed the effects of K-14585 on PAR₂-mediated [³H] inositol phosphate accumulation, MAP kinase activation, p65 NFκB phosphorylation and DNA binding and IL-8 production.

Key results:

Pretreatment with K-14585 (5 µM) inhibited [³H] inositol phosphate levels stimulated by PAR₂-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR₂-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR₂-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 µM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR₂, but not in parental or PAR₄-expressing NCTC2544 cells, suggesting these effects were PAR₂-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the G_q/11 inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR₂-mediated p65 NFκB phosphorylation and NFκB-DNA binding. K-14585 (30 µM) alone stimulated comparable NFκB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890.

Conclusions and implications:

These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR₂, one G_q/11-dependent and the other G_q/11-independent.     

Multiple muscarinic pathways mediate the suppression of voltage-gated Ca2+ channels in mouse intestinal smooth muscle cells

Background and purpose:

Stimulation of muscarinic receptors in intestinal smooth muscle cells results in suppression of voltage-gated Ca²⁺ channel currents (I_Ca). However, little is known about which receptor subtype(s) mediate this effect.

Experimental approach:

The effect of carbachol on I_Ca was studied in single intestinal myocytes from M₂ or M₃ muscarinic receptor knockout (KO) and wild-type (WT) mice.

Key results:

In M₂KO cells, carbachol (100 µM) induced a sustained I_Ca suppression as seen in WT cells. However, this suppression was significantly smaller than that seen in WT cells. Carbachol also suppressed I_Ca in M₃KO cells, but with a phasic time course. In M₂/M₃-double KO cells, carbachol had no effect on I_Ca. The extent of the suppression in WT cells was greater than the sum of the I_Ca suppressions in M₂KO and M₃KO cells, indicating that it is not a simple mixture of M₂ and M₃ receptor responses. The G_i/o inhibitor, Pertussis toxin, abolished the I_Ca suppression in M₃KO cells, but not in M₂KO cells. In contrast, the G_q/11 inhibitor YM-254890 strongly inhibited only the I_Ca suppression in M₂KO cells. Suppression of I_Ca in WT cells was markedly reduced by either Pertussis toxin or YM-254890.

Conclusion and implications:

In intestinal myocytes, M₂ receptors mediate a phasic I_Ca suppression via G_i/o proteins, while M₃ receptors mediate a sustained I_Ca suppression via G_q/11 proteins. In addition, another pathway that requires both M₂/G_i/o and M₃/G_q/11 systems may be operative in inducing a sustained I_Ca suppression.     

Cannabinoid agonists induce contractile responses through Gi/o-dependent activation of phospholipase C in the bovine ciliary muscle

Oxygen free radicals have been involved in the pathophysiology of cerebral ischemia, especially after spontaneous or thrombolytic reperfusion. In this study with rats, we have combined a severe focal ischemic insult (2 h) and a prolonged reperfusion time (7 days) to assess the possible sustained neuroprotective effect of ebselen (10 or 100 mg/kg), a small, lipophilic organoselenium compound which mimics glutathione peroxidase. Parietal cortical perfusion was measured by laser-Doppler flowmetry, and focal cerebral ischemia was carried out by the intraluminal thread method. We have measured plasma selenium levels, brain reduced glutathione levels, as a marker of oxidative stress, and infarct volume associated with cerebral ischemia. Focal ischemia did not alter reduced glutathione levels, while 60 min reperfusion following ischemia induced a significant (P < 0.05) decrease in reduced glutathione levels of the ipsilateral hemisphere. Pretreatment with ebselen, which induced significant (P < 0.05) increase in plasma selenium levels, did not significantly alter the decrease in reduced glutathione levels. The ischemic insult induced 30% mortality on average, with deaths always occurring within 12-48 h. Surviving rats suffered up to 25% body weight loss 1 week after the ischemic insult. Infarct volumes were 26.8 +/- 4.7% of the hemisphere in placebo-treated rats, 26.6 +/- 3.6% in 10 mg/kg ebselen-treated rats, and 25.6 +/- 6.4% in 100 mg/kg ebselen-treated rats (not significantly different). Single-dose administration of ebselen does not reduce the size of brain infarct resulting from severe focal cerebral ischemia in rats. In contrast to previous studies with relatively earlier endpoints, we have delayed the measurement of infarct volume to 1 week after the ischemic insult.     

S0859, an N-cyanosulphonamide inhibitor of sodium-bicarbonate cotransport in the heart

BACKGROUND AND PURPOSE: Intracellular pH (pH(i)) in heart is regulated by sarcolemmal H(+)-equivalent transporters such as Na(+)-H(+) exchange (NHE) and Na(+)-HCO(3) (-) cotransport (NBC). Inhibition of NBC influences pH(i) and can be cardioprotective in animal models of post-ischaemic reperfusion. Apart from a rabbit polyclonal NBC-antibody, a selective NBC inhibitor compound has not been studied. Compound S0859 (C(29)H(24)ClN(3)O(3)S) is a putative NBC inhibitor. Here, we provide the drug's chemical structure, test its potency and selectivity in ventricular cells and assess its suitability for experiments on cardiac contraction. EXPERIMENTAL APPROACH: pH(i) recovery from intracellular acidosis was monitored using pH-epifluorescence (SNARF-fluorophore) in guinea pig, rat and rabbit isolated ventricular myocytes. Electrically evoked cell shortening (contraction) was measured optically. With CO(2)/HCO(3) (-)-buffered superfusates containing 30 muM cariporide (to inhibit NHE), pH(i) recovery is mediated by NBC. KEY RESULTS: S0859, an N-cyanosulphonamide compound, reversibly inhibited NBC-mediated pH(i) recovery (K (i)=1.7 microM, full inhibition at approximately 30 microM). In HEPES-buffered superfusates, NHE-mediated pH(i) recovery was unaffected by 30 microM S0859. With CO(2)/HCO(3) (-) buffer, pH(i) recovery from intracellular alkalosis (mediated by Cl(-)/HCO(3) (-) and Cl(-)/OH(-) exchange) was also unaffected. Selective NBC-inhibition was not due to action on carbonic anhydrase (CA) enzymes, as 100 microM acetazolamide (a membrane-permeant CA-inhibitor) had no significant effect on NBC activity. pH(i) recovery from acidosis was associated with increased contractile-amplitude. The time course of recovery of pH(i) and contraction was slowed by S0859, confirming that NBC is a significant controller of contractility during acidosis. CONCLUSIONS AND IMPLICATIONS: Compound S0859 is a selective, high-affinity generic NBC inhibitor, potentially important for probing the transporter's functional role in heart and other tissues.     

Regulation of Fas receptor/Fas-asssociated protein with death domain apoptotic complex and associated signalling systems by cannabinoid receptors in the mouse brain

Background and purpose:

Natural and synthetic cannabinoids (CBs) induce deleterious or beneficial actions on neuronal survival. The Fas-associated protein with death domain (FADD) promotes apoptosis, and its phosphorylated form (p-FADD) mediates non-apoptotic actions. The regulation of Fas/FADD, mitochondrial apoptotic proteins and other pathways by CB receptors was investigated in the mouse brain.

Experimental approach:

Wild-type, CB₁ and CB₂ receptor knock-out (KO) mice were used to assess differences in receptor genotypes. CD1 mice were used to evaluate the effects of CB drugs on canonical apoptotic pathways and associated signalling systems. Target proteins were quantified by Western blot analysis.

Key results:

In brain regions of CB₁ receptor KO mice, Fas/FADD was reduced, but p-Ser191 FADD and the p-FADD/FADD ratio were increased. In CB₂ receptor KO mice, Fas/FADD was increased, but the p-FADD/FADD ratio was not modified. In mutant mice, cleavage of poly(ADP-ribose)-polymerase (PARP) did not indicate alterations in brain cell death. In CD1 mice, acute WIN55212-2 (CB₁ receptor agonist), but not JWH133 (CB₂ receptor agonist), inversely modulated brain FADD and p-FADD. Chronic WIN55212-2 induced FADD down-regulation and p-FADD up-regulation. Acute and chronic WIN55212-2 did not alter mitochondrial proteins or PARP cleavage. Acute, but not chronic, WIN55212-2 stimulated activation of anti-apoptotic (ERK, Akt) and pro-apoptotic (JNK, p38 kinase) pathways.

Conclusions and implications:

CB₁ receptors appear to exert a modest tonic activation of Fas/FADD complexes in brain. However, chronic CB₁ receptor stimulation decreased pro-apoptotic FADD and increased non-apoptotic p-FADD. The multifunctional protein FADD could participate in the mechanisms of neuroprotection induced by CBs.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Neurotensin excitation of serotonergic neurons in the rat nucleus raphe magnus: ionic and molecular mechanisms

To understand the cellular and molecular mechanisms by which neurotensin (NT) induces an analgesic effect in the nucleus raphe magnus (NRM), whole-cell patch-clamp recordings were performed to investigate the electrophysiological effects of NT on acutely dissociated NRM neurons. Two subtypes of neurons, primary serotonergic and secondary non-serotonergic cells, were identified from acutely isolated NRM neurons. During current-clamp recordings, NT depolarized NRM serotonergic neurons and evoked action potentials. Voltage-clamp recordings showed that NT excited serotonergic neurons by enhancing a voltage-insensitive and non-selective cationic conductance. Both SR48692, a selective antagonist of subtype 1 neurotensin receptor (NTR-1), and SR 142948A, a non-selective antagonist of NTR-1 and subtype 2 neurotensin receptor (NTR-2), failed to prevent neurotensin from exciting NRM serotonergic neurons. NT-evoked cationic current was inhibited by the intracellular administration of GDP-beta-S. NT failed to induce cationic currents after dialyzing serotonergic neurons with the anti-G(alphaq/11) antibody. Cellular Ca(2+) imaging study using fura-2 showed that NT induced the calcium release from the intracellular store. NT-evoked current was blocked after the internal perfusion of heparin, an IP(3) receptor antagonist, or BAPTA, a fast Ca(2+) chelator. It is concluded that neurotensin enhancement of the cationic conductance of NRM serotonergic neurons is mediated by a novel subtype of neurotensin receptors. The coupling mechanism via G(alphaq/11) proteins is likely to involve the generation of IP(3), and subsequent IP(3)-evoked Ca(2+) release from intracellular stores results in activating the non-selective cationic conductance.     

Differential effect of simvastatin on activation of Rac(1) vs. activation of the heat shock protein 27-mediated pathway upon oxidative stress,in human smooth muscle cells

In the present study, we have analyzed the response of human smooth muscle cell (SMC)s to oxidative stress, in terms of recruitment of key elements of the stress-activated protein kinase (SAPK) pathway, such as Rac(1), p38, and the small heat shock protein (HSP)27. The level of expression of three small HSPs, alphaB-crystallin, HSP20, HSP27, as well as the phosphorylation levels of HSP27 and p38, were higher in cultured, asynchronously growing SMCs originating from left interior mammary artery (LIMA) than those originating from aorta, saphenous vein, and umbilical vein, validating the choice of SMCs from LIMA as a model system in our study. In synchronized, quiescent SMCs from LIMA, oxidative stress (H(2)O(2) stimulation)-induced membrane translocation of Rac(1), p38 phosphorylation, membrane translocation, and phosphorylation of HSP27. In these cells, simvastatin (S), an HMG-CoA reductase inhibitor, blocked, in a mevalonate-dependent way, oxidative stress-induced membrane translocation of Rac(1). However, S pretreatment prior to oxidative stress increased the levels of p38 phosphorylation, HSP27 membrane translocation/phosphorylation, actin polymerization, and apoptosis in these cells, in a mevalonate-dependent way. These results establish that S pretreatment has a stimulatory effect on the stress-activated p38/HSP27 pathway, despite its blocking effect on Rac(1) activation.